In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detection or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To allow the acquisition of image data layer by layer, the specimen stage or the objective is displaced after a layer is scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
German Unexamined Application DE 41 11 903 A1 discloses a method for generating and correlating light-microscope images with wavelength-resolved measured data of specimens by means of single or double scanning, using a confocal scanning light microscope, of individual elements of the specimen surface to be imaged. The method comprises coupling a portion of the light out of the imaging beam path into a spectrometer, and correlating the image information with the spectroscopic data by storing the spectroscopic data in a two-dimensional region, one dimension being used for storing the measured spectrum of the individual elements, and the second dimension being activated by means of the light intensity re-emitted from the scanned elements, or by means of a criterion obtained by image processing from the overall information of the specimen image. The advantage of this method consists in the complete utilization of the capabilities of a confocal scanning light microscope and the various spectroscopic methods.
U.S. Pat. No. 6,134,002 discloses a confocal scanning microscope and a method for rapid generation of spectrally resolved images, at least two points of the specimen being scanned simultaneously.
The apparatuses and methods disclosed in the aforesaid documents have the disadvantage that the spectrum acquired in each case, which is generated from the light proceeding from the specimen, is incomplete in a very broad region of several tens of nanometers around the wavelength of the illuminating light. This is attributable to the fact that dichroic or triple-dichroic beam splitters are used on the one hand to deflect the light of a light source to the specimen and on the other hand to direct the detection light proceeding from the specimen into a detection beam path; in this context, the undesired illuminating light still present in the detection light due to scattering and reflection must be blocked out, and with dichroic or triple dichroic beam splitters that are embodied as bandpass or cutoff filters this is possible only at the cost of the information loss in the spectrum described above, since the spectral edges of the beam splitters cannot be manufactured with infinite steepness but rather have slopes that usually extend over several nanometers. Because the power level of the fluorescent light is much lower than that of the reflected excitation light, the use of semitransparent neutral splitters to separate the illuminating and detection light also does not solve the aforesaid problem, but merely causes distortion of the spectrum.
The known scanning microscopes and methods prove to be very particularly disadvantageous in applications which involve the analysis of specimens that are labeled with several dyes simultaneously, since in these experiments the illuminating light has two, three, or more wavelengths, so that the aforementioned problems are evident to an even more extreme and troublesome extent.
German Unexamined Application DE 199 06 757 discloses an optical arrangement in the beam path of a light source suitable for fluorescence excitation, preferably in the beam path of a confocal laser scanning microscope, having at least one spectrally selective element for coupling the excitation light of at least one light source into the microscope and for blocking the excitation light or excitation wavelength that is scattered and reflected at the specimen out of the light coming from the specimen via the detection beam path. For variable configuration with a very simple design, the optical arrangement is characterized in that excitation light having differing wavelengths can be blocked out by means of the spectrally selective element. Alternatively, an optical arrangement of this kind is characterized in that the spectrally selective element can be set to the excitation wavelength that is to be blocked out. The spectrally selective element can be embodied as an acoustooptical deflector (AOD) or an acoustooptical tunable filter (AOTF). In a preferred embodiment, a scanning microscope that utilizes and detects the dispersive properties of the spectrally selective element is disclosed.
This scanning microscope has the disadvantage of a very low spectral resolution, since the dispersion properties of usual acoustooptical elements, such as acoustooptical deflectors (AOD) or acoustooptical tunable filters (AOTF) are neither linear nor adequate to achieve sufficient spreading with a reasonable physical size.
The same problems occur analogously with flow cytometers, in which the specimen consists of a fluid shaped into a stream by way of a nozzle.